Method for the determination of data for the preparation of the diagnosis of phakomatosis

ABSTRACT

The invention concerns a method for the determination of data for the preparation of presymptomatic or prenatal diagnosis of phakomatosis, in particular, a tumor suppressor gene disease, in a high-risk patient, in particular of neurofibromatosis, comprising the steps of: making available the tumor material from a person afflicted with the tumor suppressor gene disease, who is a relative of the high-risk patient; isolating tumor DNA from the tumor in the relative; isolating blood DNA from the blood of the relative; amplifying polymorphous DNA microsatellite markers from the tumor and the blood; separating the markers by length; observing the lengths of the markers; comparing the markers from the blood and the tumor; examining for a loss of alleles; optionally, comparing amplified markers from a second tumor of the relative; and amplifying polymorphous DNA microsatellite markers from the blood of an offspring and separating and observing the markers.

FIELD OF THE INVENTION

[0001] The invention concerns a method for the determination of data forthe preparation of the presymptomatic or prenatal diagnosis ofphakomatosis, in particular of tumor suppressor gene diseases, inparticular of neurofibromatosis (type 1, type 2). Such methods areuseful for children of parents suffering from a hereditary disease ortheir grandchildren to increase the probability of early detection of anew occurrence of the hereditary disease, or (if possible) of prenatalevaluation.

BACKGROUND OF THE INVENTION

[0002] The current state of the art for the preparation of thecorresponding diagnosis, for this purpose, uses a mutation analysis ofthe DNA section coding for the characterizing gene for the hereditarydisease. These tumor suppressor gene diseases include the autosomaldominant inherited neurofibromatoses.

[0003] Neurofibromatosis occurs in two types, the peripheral type,called type 1, which represents approximately 85% of the cases, and the“central type,” called type 2, which represents approximately 15% of thecases. Type 1 occurs with an incidence of approximately 1:3000, whereastype 2 occurs with an incidence of approximately 1:35,000. Fordescriptions of the clinical picture, reference is made to appropriatespecialized medical books.

[0004] The drawback of the mutation analysis is that it is very timeconsuming. For example, the neurofibromtosis type 1 gene on chromosome17 (NF1 gene) has 60 exons. A complete analysis of this gene using theknown mutation analysis takes more than four months. Although theneurofibromatosis type 2 gene (NF2 gene) located on chromosome 22 issmaller, having only 17 exons, a complete analysis still takes more thanone month. In addition, one drawback of the known mutation analysis isthat in high-risk individuals the diagnosis can only be considered tohave been confirmed by molecular genetic means if a mutation is found inthe afflicted individual.

SUMMARY OF THE INVENTION

[0005] A problem of the present invention is to improve a method of thetype mentioned in the introduction. The problem is solved according tothe invention by means of a method according to the claims.

[0006] An advantage of the method according to the invention, inparticular, is that it can be carried out very quickly. Thus, in allcases, the method according to the invention can be carried out in lessthan 2 weeks; moreover, if the procedure is accelerated, it can becarried out in approximately two days. The rapidity of the methodaccording to the invention is particularly important in prenataldiagnosis. Moreover, the method according to the invention is alsoconsiderably more cost effective because of its simplicity than themutation analysis known from the state of the art.

[0007] The method according to the invention is particularlyadvantageous in cases where the known mutation analysis was unable todetect any mutation in individuals who were carriers of a mutation. Themethod according to the invention now offers the only possibility, insporadic cases, of ruling out, or confirming, neurofibromatosis of type1 or 2 on a molecular basis. In this context, the exclusion ofneurofibromatosis is of particular importance because, statistically, itis possible to rule out the disease in approximately 50% of thehigh-risk individuals. As a result, the invention not only allows theelimination of the cost-intensive mutation analyses and examinations, italso makes it possible to prevent the anxiety an individual undergoingthe examination may have concerning the possibility of having inheritedthe disease. In addition, expensive clinical examinations are also notnecessary.

[0008] In a preferred embodiment, the markers are relatively shortgene-flanking or intragenic DNA sections (to 300 bp). This offers theadvantage that material that may be available, for example in the formof paraffin blocks prepared after surgical interventions on skin tumorsin cases with neurofibromatosis, can be used, because it is possible toamplify short DNA sections from most of the available paraffin blocks. Aspecial advantage can be seen in the fact that, particularly in the caseof neurofibromatosis, the tumor material can easily be collected byexternal interventions.

[0009] In an additional preferred embodiment, at least four differentmarkers are amplified. In this manner an improved data base whichprevents possible detrimental misjudgments can be created for laterdiagnosis.

[0010] In an additional advantageous embodiment of the invention, thediagnosis of neurofibromatosis of type 1 is prepared. For this purpose,at least one polymorphous microsatellite marker from intron 27 of theNF1 gene. Furthermore, it is preferred to use at least one additionalpolymorphous microsatellite marker from intron 38 of the NF1 gene.Optimal results can be achieved when a total of three or four markersfrom the introns mentioned are used. This is advantageous because it hasbeen shown that, in a predominant number of the high-risk patientsexamined, at least one of the markers mentioned is informative. A markeris informative for a given individual if the corresponding marker ispresent in polymorphous form and having two different lengths on bothcopies of the heritable material. The markers mentioned thus guaranteethat there are two peaks in the graphic representation of the markersbased on the difference in length.

[0011] As the preparative step for the diagnosis, the physician cancompare the two peaks of the graphic representation of the markers fromthe blood of the afflicted individual, first with the result of thegraphic plotting of the length of the DNA microsatellite markers fromthe tumor, in order to establish the presence of LOH (loss ofheterozygosity=LOH). Here the invention includes the knowledge that theneurofibromas of the individuals from which the tumor material wasremoved present a 30% loss of heterozygosity, in the case of theneurofibromatosis type 1. In the case of tumors associated withneurofibromatosis type 2, the LOH rate is even higher. Thus, based onthe fact that NF1 patients present many neurofibromas, the probabilityis very high that LOH occurs in any of the neurofibromas of the patient,and thus that it is also present and can be detected in the tumormaterial made available. The LOH can be recognized in the graphicrepresentation of the markers because in the tumor material only onepeak or one imbalance of the two peaks of the corresponding marker canbe recognized. Both findings mean that the corresponding tumor has lostan allele. After the detection of LOH, the same marker from the blood ofthe high-risk person is then examined.

[0012] In another embodiment of the invention, steps c), e), g), and i)of claim 4, are repeated at least once. In this manner, a loss of anallele can again be verified or confirmed. Thus support for the firstresult can be obtained, if in the case of LOH the loss of an allele canbe confirmed in at least one of the additional examinations.

[0013] In an additional preferred embodiment such an LOH is verified, ifpossible, in at least one additional tumor of the afflicted individual,that is the above-mentioned steps are carried out with at least oneadditional tumor of the afflicted individual, if the tumor is available.In this manner the reliability of the data obtained can be furtherincreased. This is particularly advantageous in prenatal diagnosis.

[0014] An additional embodiment example of the invention is also carriedout by steps b), d), f), h) and j) of claim 4 with the blood of theparent who is not affected, if the high-risk patient is a child of bothparents. In this manner it becomes possible to determine alleles thatare not affected. This also leads, on the one hand, to an increase inthe reliability of the data obtained, and, on the other hand, in somecases, it is indispensable in the evaluation of the data obtained fordiagnosis. As an example pertaining to this, it is mentioned that it ispossible that the graphical representation of the alleles of theafflicted individual shows that he/she has alleles A and B.

[0015] In the graphical representation of the alleles of the high-riskpatient, that is in the case of the child of the afflicted individual,it is shown that the child also has alleles A and B. The graphicalrepresentation of the tumor material of the afflicted individual showsthat allele A has been lost in the tumor material of the afflictedindividual. In such a case, the data acquired would provide an unclearfoundation for a correct diagnosis, because it is unclear which one ofthe alleles A and B originates from the afflicted individual. In thiscase, in the present embodiment, the blood of the parent who was notaffected is examined. In this way a determination is made indicatingwhich alleles originate from the parent who was not affected.

[0016] If, in the present case, the parent who is not affected has theallele A or C, then it is clear the allele A could only have originatedfrom the unaffected parent. Similarly it would be clear in this examplethat allele B, which is probably exclusively responsible for the diseaseof the affected parent, was inherited by the child. In this case thechild would have an increased risk of having the disease. In the case ofNF2, it has been shown to be advantageous to use at least one of themarkers CRYB2, D22S275, NF2CA3, D22S268, D22S430.

[0017] All the data made available and graphically processed by themethod according to the invention then make it possible for thephysician who is to make the final diagnosis to evaluate whether thedisease can be ruled out in the high-risk patient examined. Indeed, ifthe examining physician then notes that the allele which was stillpresent in the tumor (as for example in the case presented below) wasnot inherited from the relative, then the occurrence of thecorresponding tumor suppressor gene disease can be ruled out.

[0018] In addition, even in the case where the high-risk patient hasinherited the allele which remained in the tumor, the physician can makea diagnosis.

[0019] In such a case two diagnostic possibilities re revealed:

[0020] i. For example, if in such a case the grandparents of thehigh-risk patient already suffer from the corresponding tumor suppressorgene disease, it can be assumed that the high-risk patient alsoinherited the disease.

[0021] ii. However, if the disease in the affected parents occurred forthe first time (sporadically), there is, on the other hand, also thepossibility that mosaic formation occurred with a probability of 20-30%,so that the genetic change in the parents suffering from the tumorsuppressor gene disease will be inherited with decreased probability bythe high-risk patient.

[0022] The method according to the invention can be used, in particular,for the preparation of presymptomatic and prenatal diagnoses ofneurofibromatosis, including NF1 and NF2 nerufibromatosis. Below, thepresent invention will be explained in an embodiment example withreference to the application of the method to high-riskneurofibromatosis patients.

[0023] European patent application EP 00113607, filed Jun. 27, 2000;Kluwe et al., (1998) “Mosaicism in Sporadic Neurofibromatosis 2Patients,” Human Molecular Genetics 7(13):2051-2055; and all otherpatents and publications cited herein are incorporated by reference.

[0024] Throughout the specification, the word “comprise,” or variationssuch as “comprises” or “comprising,” will be understood to imply theinclusion of a stated integer or group of integers but not the exclusionof any other integer or group of integers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a representation of the DNA microsatellite markersseparated according to their length, and amplified from the DNA of theblood of an afflicted individual;

[0026]FIG. 2 is a representation of the plotting by length of the samemarkers as in FIG. 1, which were amplified from the tumor material ofthe afflicted individual; and

[0027]FIG. 3 is a representation of the plotting by length of the samemarkers which were amplified from the blood of a descendant of theafflicted individual according to FIGS. 1 and 2.

[0028]FIG. 4 is a haplotype analysis of NF2 afflicted individual #358and offspring and LOH-analysis of the tumor.

EXAMPLE 1

[0029] 1. Material: Blood and Tumor Material

[0030] Tumor material from a large number of patients exist in the formof paraffin blocks prepared after surgery for the disfiguring skintumors of NF1. Tumors can be removed for cosmetic reasons at any timeand without complications. In addition, tumor material exists which hasbeen frozen and stored. All the tumors from a given patient are includedin the analysis according to the invention. The above statement inprinciple also applies to NF2, where the material usually originatesfrom neurosurgical and/or ETN interventions.

[0031] 2. DNA Isolation

[0032] The DNA is isolated from the blood or the tumor material of theaffected patient using QIAquick Blood and the QIAquick Tissue Kit fromthe company Qiagen. The procedure is described in the instructions forthe kit provided by the company.

[0033] 3. Polymorphous Markers

[0034] Four polymorphous microsatellite markers which are located inintrons 27 and 38 of the NF1 gene are amplified from the blood and tumorDNA. For each patient, at least two of these four markers should beinformative; otherwise an additional marker is used. In the case of NF2,different corresponding microsatellite markers are used (see above).

[0035] 4. Amplification of the Markers

[0036] Using primers (oligonucleotides) having a length of approximately20-24 bp, which flank the end of the DNA sections having variablelengths, the markers are amplified by PCR (Polymerase Chain Reaction).This amplification process is carried out or 10 μl of reaction solution,and it comprises blood or tumor DNA, oligonucleotides as primers, dNTPs,buffer, Taq polymerase and water. PCR is carried out in a thermocycler.For the following analysis, the primers are labeled with fluorescentdye.

[0037] 5. Analysis of the Amplified Markers

[0038] Each of the amplified markers are mixed together. To thismixture, 0.5 ROX [6-carboxy-X-rhodamine]—length standard (ABI company)and 12 μL of demineralized formamide are added. This sample is loadedafter heat denaturing onto the capillary of a Genetic Analyzer AB1310.In this separation process, the DNA sections are separated by differencein length. The results are represented graphically using the GeneScanprogram (company ABI). The DNA fragments are represented showing theirdifferent lengths and amounts.

[0039] 6. Evaluation of the Marker Analysis

[0040] If a marker in an individual is found to be informative, thereare two peaks in the graphic representation of the marker from the bloodof the person. These data are compared with the results of the analysisof the tumor material. If a tumor shows only one peak pertaining to themarker, or an imbalance in the two peaks of a marker, this means thatthe tumor has lost an allele.

[0041] For the preparation of the diagnosis, this result can then becompared with a correspondingly prepared application of the marker fromthe blood of a high-risk offspring. Furthermore, the markers from theblood of the healthy parent are correspondingly prepared.

[0042] 7. Results

[0043] To clarify the advantages of the invention, an example of aresult of the method according to the invention is explained withreference to the attached drawing.

[0044]FIG. 1 is a representation of DNA microsatellite markers separatedby length which were amplified from the DNA of the blood of an afflictedindividual. The graphic representation represents in each case thepresence of the alleles, which were called allele 2 and allele 3 in thefigure. FIG. 2 is the representation of the plotting by length of thesame markers as in FIG. 1, which were amplified from tumor material ofthe afflicted individual. One can see that the allele which was calledallele 3 in FIG. 1 is lost in the tumor of the afflicted individual.FIG. 3 is a representation of the plotting by length of the same markerswhich were amplified from the DNA of the blood of a descendant of theafflicted individual according to FIGS. 1 and 2. FIG. 3 shows that thedescendant of the afflicted individual did not inherit allele 2, whichwas still present in the tumor. Of alleles 2 and 3, the descendant onlyinherited allele 3, which was lost in the tumor. As an additionalallele, the descendant also inherited allele 1 from the other parent.For the preparation of the diagnosis of the descendant, it can thus beconcluded that the allele which is probably exclusively responsible forthe disease was not inherited by the descendant.

EXAMPLE 2

[0045] 1. Patients and Methods

[0046] An afflicted individual diagnosed as having neurofibromatosis 2(NF2) (hereinafter, “afflicted individual 358”) by the updated NIHdiagnostic criteria for NF2 was selected [Gutmann, D., et al., JAMA(1997) 278:51-57]. Biolateral vestibular schwannomas as the hallmark ofNF2. One skin schwannoma was removed from afflicted individual #358.

[0047] Methods for DNA extraction from blood from individual #358 andoffspring and tumor from individual #358 were performed as described inKluwe, et al., supra. Haplotype analysis of the afflicted individual andoffspring using allelic loss analysis of the NF2 gene in the tumor ofthe individual #358 was performed using five microsatellite markersflanking or within the NF2 gene: CRYB2, D22S275, NF2CA3, D22S268 andD22S430 [Kluwe, et al., Neurogenet. (2000) 3:17-24; Durham, I., et al.,Nature (1999) 402:489-495].

[0048] 2. Results

[0049] Afflicted individual #358 had two offspring. FIG. 4 shows thehaplotyping analysis for individual #358 and his two at-risk offspringas well as LOH-analysis for the tumor. The allele boxed with the singleline was lost in the tumor of individual #358 and inherited by offspring#358.2. The allele boxed with double line remained in the tumor ofindividual #358 and beared the mutation. This allele was inherited byoffspring #358.1.

I claim:
 1. A method for the determination of data for the preparationof presymptomatic or prenatal diagnosis of phakomatosis comprising thesteps: a. amplifying a polymorphous DNA microsatellite marker from atumor of an afflicted individual who is suffering from the phakomatosis,b. amplifying the polymorphous DNA microsatellite marker from the bloodof the afflicted individual, and c. comparing the length of theamplified polymorphous DNA microsatellite markers from steps (a) and(b).
 2. The method according to claim 1, further comprising the steps ofamplifying the polymorphous DNA microsatellite marker from the offspringof afflicted individual and comparing the length of the amplified markerwith the length of the amplified polymorphous DNA microsatellite markersfrom steps (a) and (b).
 3. The method according to claim 1 or 2, furthercomprising the steps of amplifying two or more different polymorphousDNA microsatellite markers from the tumor and the blood.
 4. A method forthe determination of data for the preparation of presymptomatic orprenatal diagnosis of phakomatosis comprising the following steps: a)making available a tumor material of an afflicted individual who issuffering from the tumor suppressor gene disease, b) making availablethe blood of the afflicted individual, c) isolating the tumor DNA fromthe tumor material, d) isolating the blood DNA from the blood, e)amplifying at least two polymorphous DNA microsatellite markers from thetumor material, f) amplifying the polymorphous DNA microsatellite markerfrom the blood, g) separating by length the polymorphous DNAmicrosatellite markers from the tumor material, h) separating by lengththe polymorphous DNA microsatellite markers from the blood, i) observingthe length of the polymorphous DNA microsatellite markers from the tumormaterial, j) observing the length of the polymorphous DNA microsatellitemarkers from the blood, and k) determining the allele which is lost inthe tumor.
 5. The method according to claim 1, further comprising thesteps of amplifying the same polymorphous DNA microsatellite marker fromthe blood of the offspring of the afflicted individual and comparing thelength of the amplified marker with the length of the amplifiedpolymorphous DNA microsatellite markers from steps (e) and (f).
 6. Themethod according to any one of claims 1-4, wherein the phakomatosis is atumor suppressor gene disease.
 7. The method according to claim 6,wherein the phakomatosis is a neurofibromatosis.
 8. The method accordingto any one of claims 1-7, where the two polymorphous markers, of whichthere are at least two, preferably have a length of up to approximately300 bp.
 9. The method according to any one of claims 1-8, wherein atleast three, or preferably four, different markers are used.
 10. Themethod according to any one of claims 1-9, wherein the marker is aneurofibromatosis gene flanking or intragenic marker.
 11. The methodaccording to claim 10, wherein at least one of the markers is located inintron 27 of the neurofibromatosis type 1 gene.
 12. The method accordingto claim 10, wherein at least one of the markers is located in intron 38of the neurofibromatosis type 1 gene.
 13. The method according to claim10, wherein, the marker is selected from the group consisting of CRYB2,D22S275, NF2CA3, D22S268 and D22S430.
 14. The method according to anyone of claims 1-7, wherein the afflicted individual is a parent of theoffspring.
 15. The method according to any one of claims 1-7, furthercomprising repeating steps d), f), h) and j).
 16. The method accordingto any one of claims 1-7, further comprising repeating steps c), e), g)and i).
 17. The method according to any one of claims 1-7, furthercomprising repeating steps c), e), g) and i) with at least oneadditional tumor of the afflicted individual.
 18. The method accordingto any one of claims 1-7, further comprising the steps of amplifying thepolymorphous DNA microsatellite marker from the blood of an unaffectedrelative of the offspring and observing the amplified marker DNA.